We have constructed various baculovirus recombinants expressing selected rotavirus proteins including VP4 (P1A, P1B, P2, P3, P4, P5B, P6 or P7), VP7 (G1, G2, G3, G4, G6 or G9) or NSP4 (genotype A, B, C or D). By using immunocytochemistry assay involving such recombinants, last year, we analyzed sera from infants who received oral attenuated rotavirus vaccine. This year, we analyzed sera obtained from gnotobiotic calves or piglets infected orally with homologous bovine (NSP4[A]) or porcine (NSP4[B]) virus. We found that following primary infection and subsequent challenge with virulent rotaviruses, naive calves and piglets developed either higher or significantly higher IgA and /or IgG antibody titers to the homologous NSP4[A] or [B] proteins than those to the heterologous rotavirus NSP4s, indicating that primary and secondary antibody responses were not NSP4 genotype-specific. Unexpectedly, such isotype antibody responses were shown to be correlated closely with the molecular phylogenetic relationships of NSP4 proteins within a species-specific region of amino acids 131-141, suggesting anti-NSP4 antibody responses were primarily species-specific. Challenge studies in piglets indicated that prior exposure to NSP4 failed to induce protection against challenge with a rotavirus that had a homologous-homotypic NSP4.